DESCRIPTION: This application proposes experiments that approach the problem of neural fate determination. The model system is a pair of neurons (s1 and s2) in the grasshopper CNS which express the neurotransmitter serotonin. s1 and s2 are sibling neurons born from the first ganglion mother cell (GMC1) of neuroblast Nb7-3. At an early stage, the entire lineage of Nb7-3 expresses the homeobox gene engrailed (en); en-expression then fades and is turned up to a high level exclusively in the serotonergic neurons at a time when these neurons have started to differentiate, more specifically, when the growth cones of these neurons approach the midline. There is good preliminary evidence showing that en-expression is causally related to later serotonin expression. The PI wants to address when en is required to induce serotonin expression, and how the surge in en-expression in s1 and s2 is regulated. The hypothesis to be tested in regard to the second aim is that environmental cues associated with the midline act upon the growth cone, which in turn signals back to the nucleus to turn up en-expression. Specific Aim 1 is technical, optimizing the technique used to block en-expression. This technique is to inject en-antisense oligonucleotides into different cells of the Nb7-3 lineage. It will be tested whether reduction in en-expression and serotonin expression can be adequately quantified, using a fluorescence microscopy/CCD camera setup. In addition, the effectiveness of en-suppression using oligos of different length, different concentration and complementarity will be established. Specific Aim 2 is to inject en-oligos into cells of the Nb7-3 lineage at different stages (neuroblast itself, GMC1, s1/s2 at different stages) and assay for expression of serotonin and other biochemical differentiation markers, as well as morphological differentiation (i.e., midline-crossing and contralateral arborization of s1/s2 axons). The hypothesis is that only late en-expression in the s1/s2 neurons themselves is essential for expressing the proper phenotype. Specific Aim 3 tests the possibility that the cues resulting in upregulation of en are picked up by the growth cone at different points along its trajectory. Favored by the P.I. is the possibility that these cues are associated with the midline cells, which are known to relay a wealth of guidance cues to axons in other systems. It is proposed to surgically transsect s1/s2 axons which had been previously filled with a fluorescent dye at different stages of their pathway, and to monitor the resulting effect on en-expression.